A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems.
A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems.
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DOI：
10.1016/j.jmb.2016.08.001
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发表时间：
2016-10-09
影响因子：
5.6
通讯作者：
Seeliger MA
中科院分区：
文献类型：
--
作者：
Lebedev I;Nemajerova A;Foda ZH;Kornaj M;Tong M;Moll UM;Seeliger MA
缺血再灌注损伤和氧化损伤导致的组织坏死是全球范围内永久性残疾和死亡的主要原因。细胞在氧化应激下发生坏死的完整机制尚不清楚。响应于氧化损伤，野生型p53已被认为是线粒体通透性转换（mPT）的中心调节组分，触发坏死。这一过程与细胞稳定和p53易位到线粒体基质中有关。在这里，我们探测p53激活关键mPT调节亲环素D（CypD）的机制。我们探讨了Trap1的参与，热休克蛋白90相关的线粒体基质蛋白和线粒体未折叠蛋白反应（mtUPR）的成员，其抑制mPT的p53依赖的方式的能力。我们的研究发现，催化活性CypD导致野生型p53蛋白（全长和分离的DNA结合结构域）在体外强烈聚集成淀粉样蛋白型原纤维。通过NMR将负责该活性的CypD残基定位于活性位点氨基酸R55、F60、F113和W121。这些数据还提出了一种新的脯氨酸异构化测定CypD通过监测聚集的p53作为CypD活性的指标。此外，我们发现，Trap1的抑制，通过特异性HSP90 ATP酶拮抗剂gamitrinib强烈敏感的原代小鼠胚胎成纤维细胞（MEFs）的mPT和通透性转换孔（mPTP）开放的p53和CypD依赖的方式。我们提出了一种机制，通过这种机制，未折叠的p53响应氧化应激而流入线粒体基质，通过将其从Trap 1复合物中取代来间接激活通常抑制的CypD。这激活CypD的异构酶活性。释放的CypD然后异构化多种蛋白质，包括p53（引起p53聚集）和mPTP孔的结构组分，诱导孔开放。这一工作模式现在可以在未来进行测试。
Tissue necrosis as a consequence of ischemia-reperfusion injury and oxidative damage is a leading cause of permanent disability and death worldwide. The complete mechanism by which cells undergo necrosis upon oxidative stress is not understood. In response to an oxidative insult, wildtype p53 has been implicated as a central regulatory component of the mitochondrial permeability transition (mPT), triggering necrosis. This process is associated with cellular stabilization and translocation of p53 into the mitochondrial matrix. Here, we probe the mechanism by which p53 activates the key mPT regulator cyclophilin D (CypD). We explore the involvement of Trap1, an Hsp90-related mitochondrial matrix protein and member of the mitochondrial unfolded protein response (mtUPR), and its ability to suppress mPT in a p53-dependent manner. Our study finds that catalytically active CypD causes strong aggregation of wildtype p53 protein (both full length and isolated DNA binding domain) into amyloid-type fibrils in vitro. The responsible CypD residues for this activity were mapped by NMR to the active site amino acids R55, F60, F113 and W121. The data also present a new proline-isomerization assay for CypD by monitoring the aggregation of p53 as an indicator of CypD activity. Moreover, we find that inhibition of Trap1 by the mitochondria-specific HSP90 ATPase antagonist gamitrinib strongly sensitizes primary mouse embryonic fibroblasts (MEFs) to mPT and permeability transition pore (mPTP) opening in a p53- and CypD-dependent manner. We propose a mechanism by which influx of unfolded p53 into the mitochondrial matrix in response to oxidative stress indirectly activates the normally inhibited CypD by displacing it from Trap1 complexes. This activates CypD’s isomerase activity. Liberated CypD then isomerizes multiple proteins including p53 (causing p53 aggregation) and the structural components of the mPTP pore, inducing pore opening. This working model can now be tested in the future.